Dissecting protein function in vivo: Engineering allelic series in mice using CRISPR-Cas9 technology

Methods Enzymol. 2022:667:775-812. doi: 10.1016/bs.mie.2022.03.053. Epub 2022 Apr 13.


Allelic series are extremely valuable genetic tools to study gene function and identify essential structural features of gene products. In mice, allelic series have been engineered using conventional gene targeting in embryonic stem cells or chemical mutagenesis. While these approaches have provided valuable information about the function of genes, they remain cumbersome. Modern approaches such as CRISPR-Cas9 technologies now allow for the precise and cost-effective generation of mouse models with specific mutations, facilitating the development of allelic series. Here, we describe procedures for the generation of three types of mutations used to dissect protein function in vivo using CRISPR-Cas9 technology. This step-by-step protocol describes the generation of missense mutations, large in-frame deletions, and insertions of genetic material using SCY1-like 1 (Scyl1) as a model gene.

Keywords: Allelic series; CRISPR; CRISPR-Cas9; Cas9; Genome editing; Mouse models; Pseudokinase; SCYL1; SCYL2; SCYL3.

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Gene Editing* / methods
  • Gene Targeting
  • Mice
  • Mutagenesis
  • Technology